A two-photon fluorescent probe with near-infrared emission for hydrogen sulfide imaging in biosystems

Article abstract of DOI:10.1039/c3cc41244j

Hydrogen sulfide (H₂S) is emerging as an important gasotransmitter but remains difficult to study. Here we report a novel two-photon fluorescent probe with NIR emission for H₂S detection. It was successfully used to realize H₂S imaging in bovine serum, living cells, tissues as well as in living mice.

Full text of DOI:10.1039/c3cc41244j

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A two-photon fluorescent probe with near-infrared
emission for hydrogen sulfide imaging in biosystems†
Cite this: Chem. Commun., 2013,
49, 3890
Wen Sun, Jiangli Fan,* Chong Hu, Jianfang Cao, Hua Zhang, Xiaoqing Xiong,
Jingyun Wang, Shuang Cui, Shiguo Sun and Xiaojun Peng*
Received 16th February 2013,
Accepted 21st March 2013
DOI: 10.1039/c3cc41244j
www.rsc.org/chemcomm
Hydrogen sulfide (H2S) is emerging as an important gasotransmitter fluorescent probes has just begun from the year 2011, there are a
but remains difficult to study. Here we report a novel two-photon few examples based on three different H2S specific reactions. They
fluorescent probe with NIR emission for H2S detection. It was made use of its reducibility, nucleophilicity and strong complexation
successfully used to realize H2S imaging in bovine serum, living ability with Cu2+, respectively.10–12 Among these reactions, it is a
cells, tissues as well as in living mice.
good choice to introduce an azido group into probes to be reduced
by H2S due to the simple synthesis, relatively good selectivity,
Hydrogen sulfide (H2S), which is believed to have been critical to the suitable reaction time, and non-cell toxicity.10
origin of life on earth, remains important in physiology and cellular
Compared with the traditional one-photon microscopy imaging
signalling.1 H2S is now considered the third member of the technique (OPM), TPM (two-photon microscopy) utilizes two-
gasotransmitter family, along with nitric oxide (NO) and carbon photons of lower energy to obtain the excited state of a fluorophore,
monoxide (CO).2 It is generated from L-cysteine in reactions and thus has more advantageous features over OPM including less
catalyzed by cystathionine-synthase (CBS) or cystathionine-lyase phototoxicity, better three dimensional spatial localization, deeper
(CSE), and converted within different organs and tissues.3 As a penetration depth and lower self-absorption.13 However, almost
signal molecule, H2S appears to be involved in various physiological all reported TP probes have short emission wavelengths ranging
processes, including modulation of neuronal transmission, regula- from 380 nm to 550 nm which limit their biological applications.
tion of release of insulin, relaxation of the smooth muscle and Therefore, it is of great value to develop a new TP fluorescent probe
reduction of the metabolic rate.4 For example, in animal models with NIR (near-infrared) emission for H2S determination which will
of critical illness, H2S donors protect from lethal hypoxia and definitely have more practical applications.
reperfusion injury and exert anti-inflammatory effects.5 The physio-
Keeping the above ideas in mind, by introducing a styrene
logically relevant H2S concentration is estimated to range from group to increase its emission wavelength to the NIR region, we
nano- to millimolar levels.6 Once the cells cannot maintain the level synthesized a conjugate extended benzopyran derivative 1-NH2,
within the physiological range, H2S is involved in diseases, such as which exhibited the desired TP absorbing property. We tested the
Alzheimer’s disease, Down syndrome, diabetes and other diseases of photophysical properties of the compound in different solvents.
mental deficiency.7 Therefore, accurate and reliable measurement of 1-NH2 showed unique spectroscopic characteristics, such as emis-
H2S concentrations in vivo is needed to provide useful information sion wavelength in the NIR region, a large Stokes shift (>100 nm in
to study the function of H2S in depth.
different solvents), good Fdmax values (50 GM at 820 nm in DMSO)
In recent years, a few analytical technologies have been developed and stable spectroscopic properties over a biologically relevant
to detect various environmental, medical and cellular H2S.8 Among pH range (Table S1 and Fig. S1a, ESI†). Upon irradiation from a
these detection technologies, fluorescence-based assays have found 500 W I-W lamp for more than 4 h, the maximal fluorescence
widespread application in the fluorescence imaging of various intensity of 1-NH2 remained nearly constant which is much better
analytes in living cells, tissues, and organisms because of the rapid, than a commercial Cy5 NIR dye in terms of photostability (Fig. S1b,
nondestructive, selective, and sensitive advantages of emission ESI†). On the basis of compound 1-NH2, we next prepared a reactive
signals.9 Actually, although the related research work on H2S H2S probe 1 by replacing the amino group with an azido group.
1 can selectively react with H2S to produce 1-NH2 as the final product
and thus result in significant fluorescence changes. Further fluores-
cence bioimaging investigations have indicated that 1 can be used as
a fluorescent probe for monitoring H2S in living cells, tissues and
State Key Laboratory of Fine Chemicals, Dalian University of Technology,
2 Linggong Road, Dalian 116024, P. R. China. E-mail: fanjl@dlut.edu.cn,
pengxj@dlut.edu.cn
in vivo in mice. To the best of our knowledge, this is the first
successful fluorescent probe for H2S detection in living mammals.
† Electronic supplementary information (ESI) available: Synthesis; experimental
details; characterization of new compounds. See DOI: 10.1039/c3cc41244j
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added, respectively, to a 5 mM solution of 1 and the fluorescence
response was monitored over 60 min. As show in Fig. 2, only H2S
could cause robust fluorescence intensity enhancement while others
exhibited almost no changes in fluorescence behaviour. The excellent
selectivity for H2S over other relative analytes shows that probe 1 has
potential applications for detection of H2S in a complex biological
environment. The pH titration curve (Fig. S1a, ESI†) also reveals that
probe 1 reaches almost a constant minimal value at pH 2.5–10,
demonstrating that 1 can work in a wide pH range without influence.
The favorable fluorescence properties of 1 for H2S prompted us to
further establish its utility for the determination of sulfide in
biosystems. 1 was first evaluated to detect H2S in commercial fetal
bovine serum. Upon addition of H2S (40 equiv.) to the serum solution
of 1 (5 mM), the fluorescence intensity increased quickly and reached
saturation after 40 min of reaction (Fig. S6a, ESI†). Bovine serum
solutions containing NaHS at different concentrations (5, 10, 20, and
40 equiv.) were also prepared. These serum solutions were then
incubated with 5 mM probe 1. After 40 min, a good linear relationship
between fluorescence intensity and NaHS concentrations was also
obtained in this solution, indicating that 1 can respond to H2S levels
in serum without addition of any co-solvents or detergents (Fig. S6b,
ESI†). Importantly, the 5 mM concentration of 1 used in this
experiment was much lower than that of other reported H2S probes
(100–200 mM).10a,11a,b The low concentration of the probe will be able
to greatly reduce the damage to biological systems.
We next acquired fluorescence imaging of H2S in living cells
utilizing an Olympus FV1000-IX81 confocal microscope. Using
800 nm TP excitation in a scanning lambda mode, MCF-7 cells
incubated with only probe 1 (5 mM) for 30 min at 37 1C in PBS
exhibited weak fluorescence at an emission window of 575–630 nm
(Fig. 3a), suggesting that 1 is immune to metabolism of various
intracellular species under the assay conditions. Whereas addition
of H2S (50 equiv.) to the above cells resulted in a distinct change in
the observed fluorescence after incubation for another 60 min
(Fig. 3c). Photo imaging of H2S in HeLa cells was also successfully
realized with excitation at 515 nm (Fig. S8, ESI†). The effect of a
hypoxic atmosphere on the probe was also examined according to
the reported method.14 Although hypoxia could cause low-level
fluorescence intensity in living cells (Fig. S10, ESI†), the effect can
be effectively avoided when performing an in vivo experiment. These
data indicate that probe 1 is cell membrane permeable and capable
Scheme 1 Synthesis of probe 1 and the proposed mechanism of response of
1 to hydrogen sulfide. (i) Ethyl pyruvate with Na in CH3COOC2H5, 4 h, 53%;
(ii) AcOH, H2SO4, 30 min, 76.9%; (iii) malononitrile, AcOH, H2SO4, 14 h, 32.5%;
(iv) toluene, piperidine, AcOH, 3 h, 42.9%.
The benzopyran derivative 1 (Scheme 1) was prepared from
1-(2-hydroxyphenyl)ethanone by a five-step procedure under
mild conditions with a good yield. The structures of 1 and
other major intermediates were well characterized using
1H NMR, 13C NMR, and HPLC-MS (see ESI†).
We then tested the optical properties of probe 1 in PBS buffer
(0.1 M, pH 7.4, 50% DMSO). UV-vis spectra of 1 (5 mM) exhibited two
absorptions at around 400 nm and 425 nm. After treatment with
50 equiv. of NaHS (a commonly used hydrogen sulfide source) at
37 1C, the absorption at 400 nm and 425 nm apparently decreased,
whereas a new absorption peak appeared at about 505 nm. Such a
large red shift of 90 nm in the absorption behavior changed the
color of the solution from yellow to orange red, allowing colorimetric
detection of H2S by the naked eye (Fig. S2, ESI†). Accordingly, the
emission at 670 nm strongly appeared upon excitation at 520 nm
(Fig. 1a), which means that the azido group of 1 can be converted
efficiently into fluorescent 1-NH2. Within 60 min of reaction under
these conditions, a 65-fold turn-on response of the fluorescent signal
was observed (Fig. S4, ESI†). Different concentrations of NaHS were
then added to the test solution, and the fluorescence intensity
increased linearly with the concentration of H2S from 25 mM up to
250 mM (Fig. 1b and S5, ESI†). Thus, the detection limit (3s/slope)
was as low as 3.05 mM.
The turn-on response of probe 1 for H2S was tested over reactive
oxygen species (ROS), biologically relevant sulfide species (RSS) and
reducing reagents including hypochlorite (ClOÀ), sulfite (SO32À),
bisulfite (HSO3À), thiosulfate (S2O32À), dithionite (S2O42À), cysteine
(Cys), homocysteine (Hcy), glutathione (GSH), IÀ, Fe2+ and Cu+. In the
test system, 50 equiv. of H2S and 100 equiv. of other analytes were
Fig. 1 (a) Fluorescence spectra of 1 (5 mM) and 1-NH2 (5 mM) in PBS buffer
(0.1 M, pH 7.4, 50% DMSO). (b) Fluorescence change in 1 (5 mM) after incubation
with different concentrations of H2S (5, 10, 20, 30, 40 and 50 equiv.) for 60 min.
Conditions: PBS buffer (0.1 M, pH 7.4, 50% DMSO) at 37 1C, lex = 520 nm,
each spectrum was obtained after 60 min of reaction.
Fig. 2 Fluorescence responses of probe 1 (5 mM) to biologically relevant ROS,
RSS and reducing reagents. Bars represent relative responses at 670 nm at 0, 5,
10, 20, 40 and 60 min after addition of analytes. Data are shown for 50 equiv. of
H2S and 100 equiv. of other analytes. Conditions: PBS buffer (0.1 M, pH 7.4, 50%
DMSO) at 37 1C, lex = 520 nm.
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established that 1 is a desired imaging agent used for visualizing
H2S in vivo for the first time.
In summary, we presented a new fluorescent probe 1 for specific
H2S detection with two-photon absorption and NIR emission. It
exhibited a significant fluorescence response for H2S over other
reactive oxygen species and biologically relevant reactive sulfide
species. Thus it is suitable for H2S detection in bovine serum with a
quite low probe concentration. Living cells and tissue imaging
Fig. 3 Fluorescence imaging of H2S in MCF-7 cells incubated with 5 mM probe 1.
Cells were incubated with 1 for 30 min (a), after which 50 equiv. of H2S was
added. After further incubation for 30 min (b) and 60 min (c) the cells were established the utility of this probe for tracking H2S in biological
imaged. lex = 800 nm, emission window (575 nm–630 nm). Scale bars: 20 mm.
systems using TPM microscopy. More importantly, the probe was
first successfully utilized to realize H2S imaging in living mice.
of imaging H2S in different living cells by using both OPM and TPM.
Cytotoxicity test using MCF-7 cells showed that probe 1 and the
corresponding product 1-NH2 have almost no toxicity to living cells
(Fig. S11, ESI†).
Taken together, 1 is a robust imaging agent for H2S detection both
in vitro and in vivo. We expect this new probe to be useful in more
chemical and biological applications.
This work was supported by NSF of China (21136002,
20923006 and 21076032) and National Basic Research Program
of China (2013CB733702 and 2013CB733702).
We further investigated the utility of probe 1 in tissue imaging.
Since it takes a longer time to stain the tissues during which they
may be deformed, an excess amount (20 mM) of 1 was used to
facilitate staining. A fresh rat liver cancer slice incubated with 20 mM
probe 1 for 60 min showed weak fluorescence in the whole region.
After incubation with H2S (50 equiv.) for another 60 min, there was
an even and significant increase in fluorescence intensity excited at
800 nm (Fig. S12, ESI†). Furthermore, depth scanning demonstrated
that the corresponding product 1-NH2 was capable of tissue imaging
at depths of 60–220 mm by TPM (Fig. S13, ESI†).
Finally, we examined the suitability of the sensor for visualizing
H2S in living animals. ICR mice were selected and divided into two
groups. One group was given an s.p. (skin-pop) injection of probe 1
(40 mM, in 25 mL DMSO) on the back of ICR mice as the control
experiment. The other group was then given an s.p. injection of
25 equiv. of NaHS (25 mL, 0.1 mM PBS) after the disposal of the
control mice. The two groups were imaged using a NightOWL II
LB983 small animal in vivo imaging system with a 530 nm excitation
laser and a 655 Æ 20 nm emission filter. Fig. 4 shows a representa-
tive fluorescent image of mice treated with probe 1 and NaHS at
different times after injection and demonstrates that the fluores-
cence intensity became strong gradually within 4 hours. Whereas
the control experiment shows almost no fluorescence, proving that
probe 1 can detect H2S in vivo without the interference of back-
ground signals (Fig. S14a, ESI†). Taken together these experiments
Notes and references
1 B. K. Czyzewski and D.-N. Wang, Nature, 2012, 483, 494–498.
2 (a) L. Li, P. Rose and P. K. Moore, Annu. Rev. Pharmacol. Toxicol.,
2011, 51, 169–187; (b) M. M. Gadalla and S. H. Snyder, J. Neurochem.,
2010, 113, 14–26.
3 B. D. Paul and S. H. Snyder, Nat. Rev. Mol. Cell Biol., 2012, 13,
499–507.
4 (a) H. Kimura, Amino Acids, 2011, 41, 113–121; (b) E. Blackstone,
M. Morrison and M. B. Roth, Science, 2005, 308, 518.
5 J. L. Wallace, Trends Pharmacol. Sci., 2007, 28, 501–504.
6 Y. Han, J. Qin, X. Chang, Z. Yang and J. Du, Cell. Mol. Neurobiol.,
2006, 26, 101–107.
7 (a) K. Eto, T. Asada, K. Arima, T. Makifuchi and H. Kimura, Biochem.
Biophys. Res. Commun., 2002, 293, 1485–1488; (b) P. Kamoun,
M.-C. Belardinelli, A. Chabli, K. Lallouchi and B. Chadefaux-
Vekemans, Am. J. Med. Genet., Part A, 2003, 116, 310–311;
(c) W. Yang, G. Yang, X. Jia, L. Wu and R. Wang, J. Physiol., 2005,
569, 519–531.
8 (a) S. K. Pandey and K.-H. Kim, Environ. Sci. Technol., 2009, 43,
3020–3029; (b) B. K. Reese, D. W. Finneran, H. J. Mills, M.-X. Zhu
and J. W. Morse, Aquat. Geochem., 2011, 17, 567–582.
9 (a) M. E. Jun, B. Roy and K. H. Ahn, Chem. Commun., 2011, 47,
7583–7601; (b) X. J. Zhao and C. Z. Huang, TrAC, Trends Anal. Chem.,
´
´
2010, 29, 354–367; (c) M. Fernandez-Suarez and A. Y. Ting, Nat. Rev.
Mol. Cell Biol., 2008, 9, 929–943; (d) C. Chung, D. Srikun, C. S. Lim,
C. J. Chang and B. R. Cho, Chem. Commun., 2011, 47, 9618–9620.
10 (a) H. Peng, Y. Cheng, C. Dai, A. L. King, B. L. Predmore, D. J. Lefer
and B. Wang, Angew. Chem., Int. Ed., 2011, 50, 1–5; (b) L. A. Montoya
and M. D. Pluth, Chem. Commun., 2012, 48, 4767–4769; (c) S. K. Das,
C. S. Lim, S. Y. Yang, J. H. Han and B. R. Cho, Chem. Commun., 2012,
48, 8395–8397; (d) S. Chen, Z.-j. Chen, W. Ren and H.-w. Ai, J. Am.
Chem. Soc., 2012, 134, 9589–9592; (e) A. R. Lippert, E. J. New and
C. J. Chang, J. Am. Chem. Soc., 2011, 133, 10078–11080; ( f ) F. Yu,
P. Li, P. Song, B. Wang, J. Zhao and K. Han, Chem. Commun., 2012,
48, 2852–2854; (g) Q. Wan, Y. Song, Z. Li, X. Gao and H. Ma, Chem.
Commun., 2012, 48, 502–504.
11 (a) C. Liu, J. Pan, S. Li, Y. Zhao, L. Y. Wu, C. E. Berkman,
A. R. Whorton and M. Xian, Angew. Chem., Int. Ed., 2011, 123,
10511–10513; (b) Y. Qian, L. Zhang, S. Deng, C. He and J. Zhao,
Chem. Sci., 2012, 3, 2920–2922; (c) X. Cao, W. Lin, K. Zheng and
L. He, Chem. Commun., 2012, 48, 10529–10531.
12 K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura,
T. Komatsu, T. Ueno, T. Terai, H. Kimura and T. Nagano, J. Am.
Chem. Soc., 2011, 133, 18003–18005.
13 (a) M. Pawlicki, H. A. Collins, R. G. Denning and H. L. Anderson,
Angew. Chem., Int. Ed., 2009, 48, 3244–3266; (b) H. M. Kim and
B. R. Cho, Acc. Chem. Res., 2009, 42, 863–872; (c) T. Terai and
T. Nagano, Curr. Opin. Chem. Biol., 2008, 12, 515–521.
14 S. Takahashi, W. Piao, Y. Matsumura, T. Komatsu, T. Ueno, T. Terai,
T. Kamachi, M. Kohno, T. Nagano and K. Hanaoka, J. Am. Chem.
Soc., 2012, 134, 19588–19591.
Fig. 4 Representative fluorescence images of mice (pseudo-color) given an s.p.
injection of probe 1 (40 mM, in 25 mL DMSO) and then injected with 25 equiv. of
NaHS (25 mL, 0.1 mM PBS). Images were taken after incubation of NaHS for
different times (0, 0.5, 1, 2, 3 and 4 h).
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3892 Chem. Commun., 2013, 49, 3890--3892
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